Thermodynamic equilibrium is a fundamental and inherent feature of reactions catalyzed by isomerase enzymes. During industrial production of chemicals of interest involving isomerization reactions, this feature always leads to not only incomplete conversion of upstream substrates, but also difficulties and extra cost on downstream product separation and purification. One typical example is the rare sugar industry which by far has been using isomerization reactions (1-4) and thus cannot outcompete other sweetener industries due to the significantly higher production cost, even though rare sugars exhibit numerous beneficial traits. Specifically, tagatose, a naturally occurring functional sweetener with 92% of the sweetness as sucrose in 10% (w/w) solution, but with only 1.5 kcal/g compared with table sugar's 4 kcal/g (1, 2), has been widely produced through isomerization reaction from galactose. Its conversion rate is always low due to the thermodynamic equilibrium problems (3, 4). For example, the thermodynamic equilibrium between galactose and tagatose is 7:3 based on L-arabinose isomerase (L-AI) reaction (5, 6). Maximum conversion rate of 35% for L-AI from Lactobacillus reuteri was achieved by increasing the temperature to 60° C. (7). The bioconversion yield of galactose to tagatose by the purified thermostable L-AI from Bacillus stearothermophilus reached 36% after 12 h at 65° C. (8). The conversion rate of 68% was achieved using L-AI from Thermotoga neapolitana at 80° C. (9). The addition of boric acid increased the conversion rate to 74% using L-AI mutant enzyme purified from Geobacillus thermodenitrificans at 60° C. (10).
As such, although galactose-tagatose conversion rate can be improved when high temperature is used, it is not cost-effective and still cannot achieve complete substrate conversion. In addition, to obtain galactose, the direct substrate for tagatose production, an enzymatic hydrolysis of lactose and a follow-up separation of glucose and galactose are required, leading to substantial extra cost. Moreover, the industrial scale-up cost increases sharply when purified enzymes instead of a self-sustaining bioconversion system are used. Therefore, the current tagatose has industry encountered a bottleneck on reducing production cost. Cost effective methods of producing rare sugars via fermentation are needed in the art.